Methods of use of soluble cd24 for therapy of rheumatoid arthritis

ABSTRACT

Provided herein is a CD24 protein. The CD24 protein may include mature human or mouse CD24, as well as a N- or C-terminally fused portion of a mammalian immunoglobulin.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a continuation-in-part of U.S. patent application Ser. No.13/643,527, which is the national stage of International Application No.PCT/US11/34282, filed Apr. 28, 2011, which claims the benefit of U.S.Provisional Patent Application No. 61/329,078 filed on Apr. 28, 2010,the contents of all which are incorporated herein by reference.

FIELD OF THE INVENTION

This invention relates to compositions and methods for treatingrheumatoid arthritis.

BACKGROUND OF THE INVENTION

This section provides background information which is not necessarilyprior art and a general summary of the present disclosure which is not acomprehensive disclosure of its full scope or all of its features.

CD24 is known as the heat-stable antigen. It is expressed as aglycosyl-phosphatidyl-inositol (GPI)-anchored molecule and has a widedistribution in different lineages. Because of the tendency of CD24 tobe expressed on immature cells, it has also been used as part of stemcell markers and for lymphocyte differentiation. The first functionassociated with CD24 is a costimulatory activity for antigen-specific Tcell response. In vivo studies indicated that, as a costimulator for Tcell activation in the lymphoid organ, CD24 is redundant but becomesessential in the absence of CD28. This would not be the case for localtarget organs that are not as “costimulator rich.” Consistent with thisnotion, it has been demonstrated that mice with a targeted mutation ofCD24 are completely resistant to induction of experimental autoimmuneencephalomyelitis (EAE).

Polymorphisms of human CD24 are associated with risk and progression ofseveral autoimmune diseases, including multiple sclerosis and rheumatoidarthritis (RA). In cases of multiple sclerosis, it has been reportedthat soluble CD24, consisting of the extracellular portion of murineCD24 and human IgG1 Fc ameliorated the clinical symptom of experimentalautoimmune diseases, the mouse model of multiple sclerosis. More recentstudies have demonstrated that CD24 interact with and represses hostresponse to danger-associated molecular patterns (DAMPs).

RA affects 0.5-1% of human populations. Although a number ofdisease-modifying antirheumatic drugs (DMARDs) are currently available,even the gold standard of biologic DMARDs, the therapeutics targetingthe tumor-necrosis factor alpha, lead to 50% improvement according toAmerican College of Rheumatology Improvement Criteria (ACR50) in lessthan 50% of the patients receiving the treatments. No cure for RA isavailable. It is therefore necessary to test additional therapeutics forRA. RA is presumed to be autoimmune diseases in the joint, although thecause of the diseases remains largely obscure. A number of studies haveimplicated T cells in the pathogenesis of rheumatoid arthritis. Morerecently, it has been demonstrated that transfer of antibodies can causethe development of inflammation of the joints of mice. The pathology ofthe lesions resembles human rheumatoid arthritis.

Animal models relevant to human RA played an important role for theadvancement of therapeutic development in DMARDs. For example,collagen-induced arthritis in the mouse and rat were critical for thedevelopment of therapeutics for RA. More recently, it has beendemonstrated that adaptive transfer of anti-collagen antibodies causerobust RA-like lesion in the mice. Since auto-antibodies are elevated inRA patients prior to the onset of diseases, passive transfer ofcollagen-specific antibody is a relevant model for human RA.

Since the pathogenesis of RA involves host response to DAMP and sincethe CD24 molecule negatively regulate host response to DAMPs, thepotential of using soluble CD24 to treat RA was investigated. Thepassive transfer model of RA was chosen because of both relevance tohuman diseases and simplicity of experimental designs.

SUMMARY OF THE INVENTION

Provided herein is a CD24 protein comprising a mature human CD24 variantconsisting of SEQ ID NO: 1. The CD24 protein may further comprise aportion of a mammalian immunoglobulin (Ig), which may be fused to theN-terminus or C-terminus of the mature CD24. The Ig portion may be theFc portion of a human Ig protein. The Fc portion may consist of thehinge region and CH2 and CH3 domains of the human Ig protein, and the Igmay be IgG1, IgG2, IgG3, IgG4, or IgA. The Fc portion may consist of thehinge region and CH3 and CH4 regions of IgM.

The CD24 protein may be soluble, and may be glycosylated. The CD24protein may also be produced using a eukaryotic protein expressionsystem, which may comprise a vector contained in a Chinese Hamster Ovarycell line or a replication-defective retroviral vector. Thereplication-defective retroviral vector may be stably integrated intothe genome of a eukaryotic cell.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-B. FIG. 1A shows the amino acid composition of the CD24 fusionprotein, CD24IgG1Fc (also referred to herein as CD24Fc) (SEQ ID NO: 5).The underlined 26 amino acids are the signal peptide of CD24 (SEQ ID NO:4). The boxed, bold portion of the sequence is the mature CD24 proteinused in the fusion protein (SEQ ID NO: 1). The last amino acid (A or V)that is ordinarily present in the mature CD24 protein has been deletedfrom the construct to avoid immunogenicity. The non-underlined, non-boldletters are the sequence of IgG1 Fc, including the hinge region and CH1and CH2 domains (SEQ ID NO: 6). FIG. 1B shows the sequence of CD24^(v)Fc(SEQ ID NO: 7), in which the mature human CD24 protein is the valinepolymorphic variant of SEQ ID NO: 2. The various parts of the fusionprotein are marked as in FIG. 1A.

FIG. 2. Methods for purification and processing of CD24IgG1Fc (CD24Fc)expressed from mammalian cell lines.

FIG. 3. Amino acid sequence variations between mature CD24 proteins frommouse (SEQ ID NO: 3) and human (SEQ ID NO: 2). The potentialglycosylation sites are bolded, with the N-glycosylation sites in red.

FIGS. 4A-C. WinNonlin compartmental modeling analysis ofpharmacokenitics of CD24IgG1 (CD24Fc). The opened circles represent theaverage of 3 mice, and the line is the predicted pharmacokinetic curve.FIG. 4A. i.v. injection of 1 mg CD24IgG1. FIG. 4B. s.c. injection of 1mg CD24IgG1 (CD24Fc). FIG. 4C. Comparison of the total amounts ofantibody in the blood as measured by areas under curve (AUC), half-lifeand maximal blood concentration. Note that overall, the AUC and Cmax ofthe s.c. injection is about 80% of i.v. injection, although thedifference is not statistically significant.

FIGS. 5A-B. CD24-Siglec G (10) interaction discriminates between PAMPand DAMP. FIG. 5A. Host response to PAMP was unaffected by CD24-SiglecG(10) interaction. FIG. 5B. CD24-Siglec G (10) interaction represseshost response to DAMP, possibly through the Siglec G/10-associatedSHP-1.

FIGS. 6A-B. A single injection of CD24Fc reduces clinical score of CAIA.FIG. 6A. Diagram of experiments. BALB/c mice (8 weeks old) received mAbson day 1 in conjunction with either vehicle or fusion proteins. The micewere injected LPS on day 3, and were observed daily for 3 weeks. FIG.6B. CD24Fc reduces clinical scores of CAIA. The fusion proteins (1mg/mouse) or vehicles were injected once on day 1. Clinical scores weredetermined double blind. *, P<0.05; **, P<0.01; ***, P<0.001. The effectof CD24 was reproduced in 6 independent experiments, involving a totalof 52 mice in the PBS group and 54 mice in CD24Fc group.

FIGS. 7A-B. CD24Fc reduces the levels of inflammatory cytokines in thejoint and CAIA. CAIA initiated and treated as diagramed in FIG. 6A. Theinflammatory cytokines were measured by cytokine bead array from BDPharmingen. FIG. 7A. Representative FACS profile. FIG. 7B. The summaryof reduced cytokines (Mean±SE) measured in the joint homogenates.

FIG. 8. CD24Fc reduces inflammation and destruction of cartilage in thejoint. On day 7, front and hind paws were dissected from both CD24Fctreated and control mice, fixed in 4% paraformaldehyde for 24 hoursfollowed by decalcification with 5% formic acid. The paws were thenembedded in paraffin and the longitudinal section were stained with H&Eand Safranin 0 red (Sigma-Aldrich).

FIG. 9. Therapeutic effect of CD24Fc administrated on day 5 of CAIAinduction. The CAIA-induced mice were randomized into two groups,receiving either vehicle (PBS) or CD24 Fc. The mice were scored doubleblind. Representative of three independent experiments are shown.

FIGS. 10A-B. Low doses of CD24Fc prevent development of CAIA. FIG. 10A.Diagram of experiments. FIG. 10B. Clinical scores of arthritis, scoreddouble blind.

FIGS. 11A-B. Siglecg is essential for therapeutic effect of CD24Fc, WT(FIG. 11A) and Siglec^(−/−) mice (FIG. 11B) received either vehiclecontrol or CD24Fc in conjunction of a cocktail of anti-collagen mAbs.The clinical scores were recorded daily double blind.

FIGS. 12A-F. Construction of CD24^(v)Fc and CD24Fc. FIG. 12A. Diagram ofthe fusion proteins. The polymorphic residue in extracellular domain wasdeleted in CD24Fc. FIG. 12B. SDS-PAGE analysis for the purity of the twofusion proteins. The numbers shown are μg of proteins loaded. FIG. 12C.Comparison between CD24^(v)Fc and CD24Fc for their binding toSiglec10Fc. Desialylated CD24Fc was used as a negative control. FIG.12D. Comparison between CD24Fc and CD24^(v)Fc for the therapeutic effectin the CAIA model. CD24Fc or CD24^(v)Fc (200 μg/mouse) was injected intomice in conjunction with a cocktail of anti-collagen antibodies onday 1. Arthritis was elicited by treatment with LPS on day 3. Thediseases were scored double blind. Data shown in FIGS. 12C and D aremeans and SEM. FIGS. 12E and 12F also compare the therapeutic effects ofCD24Fc and CD24^(v)Fc, in experiments performed similarly to the onesshown in FIG. 12D, except that IgG1 Fc was used as a negative control.As shown in FIG. 12E, CD24Fc reduced the RA score as early as day 4, andshowed statistically significant protection throughout the three weeksof observation. On the other hand, as shown in FIG. 12F, CD24^(v)Fcshowed a reduction in RA score starting on day 8. Although reducedscores were observed thereafter, the reduction did not reach statisticalsignificance.

FIGS. 13A-B. CD24Fc conferred protection against CIA in DBA/1 mice. FIG.13A. CD24Fc suppressed development of arthritis in the CIA model. Micereceived a single treatment (1 mg/mouse) on day 17 when no clinicalsymptoms had developed. Data shown are disease scores among the micethat had developed arthritis with disease scores from 3 to 8. Thedifference between CD24Fc and PBS group was significant (P=0.02,Fisher's PLSD test). N=9 for vehicle and N=7 for CD24Fc group. FIG. 13B.Therapeutic effect of CD24Fc in CIA of DBA/1 mice. The mice withclinical symptoms of scores from 3 to 8 were randomized to receiveeither 200 μg CD24Fc or an equal volume of control vehicle (PBS) by i.p.injection, every the other day, five times. The mice were inspecteddaily to score for the clinical symptoms for two weeks. CD24Fcsignificantly lessened the clinical symptoms of arthritis when arthritisdeveloped (P=0.02, Fisher's PLSD test). N=6 for PBS and N=5 for CD24Fcgroups.

FIGS. 14A-B. CD24Fc caused rapid recovery in mice with ongoing chickenCIA. On day 1, 8-week old C57BL/6 mice were immunized with 100 μL ofcollagen-CFA emulsion (made by mixing 4 mg/ml of chick type II collagenwith equal volume of CFA containing 5 mg/ml of M. tuberculosis)intradermally at the base of the tail. On day 21, booster immunizationwith the same collagen-CFA emulsion was administered intradermally 1.5cm from the tail base. FIG. 14A. On day 28, mice with a clinicalscore >3 were randomized to receive either vehicle or CD24Fc (1mg/mouse). The endpoint was a reduction of score by 50% (top) or 80%(bottom). N=12 for PBS, and N=11 for CD24Fc. FIG. 14B. Dose-dependenttherapeutic effect of CD24Fc in chicken CIA model. Details as in FIG.14A, except that the treatments started at the peak of disease (averagescore of 5.5 in both groups on day 33). Mice with a clinical score >3were randomized to receive 5 injections of either vehicle or CD24Fc. Theendpoint was a reduction of score by either 50% (top) or 80% (bottom).N=11. The difference between the 100 μg experimental and the vehiclecontrol groups was statistically significant.

FIGS. 15A-C. CD24 inhibited inflammatory cytokine production by humanmacrophages. FIG. 15A. ShRNA silencing of CD24 led to spontaneousproduction of TNFα, IL-6 and IL-1β. THP1 cells were transduced withlentiviral vectors encoding either scrambled or two independent CD24shRNA. The transduced cells were differentiated into macrophages byculturing for 4 days with PMA (15 ng/ml). After washing away PMA andnonadherent cells, the cells were cultured for another 24 hours formeasurement of inflammatory cytokines by cytokine beads array. FIG. 15B.As in FIG. 15A, except that the given concentration of CD24Fc or controlIgG Fc was added to macrophages in the last 24 hours. FIG. 15C. CD24Fcwas more efficient than CD24^(v)Fc in suppressing the spontaneousproduction of inflammatory cytokines by CD24-silenced macrophage cellline THP1. The data shown are as detailed in the FIG. 12 legends, exceptthat the CD24Fc and CD24^(v)Fc are compared side-by-side.

FIGS. 16A-C. Contribution of Siglec G to protection by CD24Fc. FIG. 16A.CD24Fc stimulated tyrosine phosphorylation of, and SHP-1 binding to,Siglec G. Spleen cells from CD24-deficient mice were stimulated witheither vehicle, Fc control or CD24Fc (1 μg/ml) for 30 min. After lysis,the Siglec G protein was precipitated with anti-Siglec G antisera.Siglec G phosphorylation and its association to SHP-1 were detected byWestern blot. FIG. 16B. Siglecg was essential for therapeutic effect ofCD24Fc in mice with low dose of anti-collagen antibodies. WT (FIG. 16A)and Siglec^(−/−) mice (FIG. 16B) received either vehicle control orCD24Fc in conjunction of a cocktail of anti-collagen mAbs (2 mg/mouse).LPS was injected on day 3 (100 μg/mouse). The clinical scores wererecorded daily double blind. Data are representative of two experiments.FIG. 16C. Targeted mutation of Siglecg attenuated but did not abrogatethe therapeutic effect of CD24Fc with double doses of anti-collagenantibodies. The anti-collagen antibodies (4 mg/mouse) and CD24Fc (1mg/mouse) were added on day 1, while LPS (100 μg/mouse) was added on day3. Male WT (FIG. 16A) and Siglec^(−/−) mice (FIG. 16B) were observeddaily for clinical score. % inhibitions were calculated by % reductionof accumulated RA score. N=5. Male mice were used at 8 weeks of age.

FIGS. 17A-B. FIGS. 17A and B show pharmacokinetic profiles of CD24 inmale and female mice at doses of 12.5, 35, and 125 mg/kg.

FIGS. 18A and B show CD24Fc serum concentrations vs. time in cynomolgusmonkeys (12.5 mg/kg dose).

DETAILED DESCRIPTION

The inventors have discovered that a soluble form of CD24 is highlyeffective for treating rheumatoid arthritis. In particular, theinventors have discovered that a variant CD24 fusion protein in whichthe core of human CD24 lacks the polymorphic amino acid at position 57of full-length CD24 has a superior therapeutic effect when compared witha CD24 protein which has a wild-type core CD24 sequence.

1. DEFINITIONS

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting. As used in thespecification and the appended claims, the singular forms “a,” “an” and“the” include plural referents unless the context clearly dictatesotherwise.

For recitation of numeric ranges herein, each intervening number therebetween with the same degree of precision is explicitly contemplated.For example, for the range of 6-9, the numbers 7 and 8 are contemplatedin addition to 6 and 9, and for the range 6.0-7.0, the numbers 6.0, 6.1,6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitlycontemplated.

A “peptide” or “polypeptide” is a linked sequence of amino acids and maybe natural, synthetic, or a modification or combination of natural andsynthetic.

“Substantially identical” may mean that a first and second amino acidsequence are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,98%, or 99% over a region of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150,160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or300 amino acids.

“Treatment” or “treating,” when referring to protection of an animalfrom a disease, means preventing, suppressing, repressing, or completelyeliminating the disease. Preventing the disease involves administering acomposition of the present invention to an animal prior to onset of thedisease. Suppressing the disease involves administering a composition ofthe present invention to an animal after induction of the disease butbefore its clinical appearance. Repressing the disease involvesadministering a composition of the present invention to an animal afterclinical appearance of the disease.

A “variant” may mean means a peptide or polypeptide that differs inamino acid sequence by the insertion, deletion, or conservativesubstitution of amino acids, but retain at least one biologicalactivity. Representative examples of “biological activity” include theability to bind to a toll-like receptor and to be bound by a specificantibody. Variant may also mean a protein with an amino acid sequencethat is substantially identical to a referenced protein with an aminoacid sequence that retains at least one biological activity. Aconservative substitution of an amino acid, i.e., replacing an aminoacid with a different amino acid of similar properties (e.g.,hydrophilicity, degree and distribution of charged regions) isrecognized in the art as typically involving a minor change. These minorchanges can be identified, in part, by considering the hydropathic indexof amino acids, as understood in the art. Kyte et al., J. Mol. Biol.157:105-132 (1982). The hydropathic index of an amino acid is based on aconsideration of its hydrophobicity and charge. It is known in the artthat amino acids of similar hydropathic indexes can be substituted andstill retain protein function. In one aspect, amino acids havinghydropathic indexes of ±2 are substituted. The hydrophilicity of aminoacids can also be used to reveal substitutions that would result inproteins retaining biological function. A consideration of thehydrophilicity of amino acids in the context of a peptide permitscalculation of the greatest local average hydrophilicity of thatpeptide, a useful measure that has been reported to correlate well withantigenicity and immunogenicity. U.S. Pat. No. 4,554,101, incorporatedfully herein by reference. Substitution of amino acids having similarhydrophilicity values can result in peptides retaining biologicalactivity, for example immunogenicity, as is understood in the art.Substitutions may be performed with amino acids having hydrophilicityvalues within ±2 of each other. Both the hyrophobicity index and thehydrophilicity value of amino acids are influenced by the particularside chain of that amino acid. Consistent with that observation, aminoacid substitutions that are compatible with biological function areunderstood to depend on the relative similarity of the amino acids, andparticularly the side chains of those amino acids, as revealed by thehydrophobicity, hydrophilicity, charge, size, and other properties.

2. CD24

Provided herein is a CD24 protein, which may have the amino sequence ofmature human CD24, which may be SETTTGTSSNSSQSTSNSGLAPNPTNATTK (SEQ IDNO: 1) or SETTTGTSSNSSQSTSNSGLAPNPTNATTK(V/A) (SEQ ID NO: 2), or mouseCD24, which may be NQTSVAPFPGNQNISASPNPTNATTRG (SEQ ID NO: 3), or avariant thereof. The CD24 may be soluble. The CD24 may further comprisea N-terminal signal peptide, which may have the amino acid sequenceMGRAMVARLGLGLLLLALLLPTQIYS (SEQ ID NO: 4). The CD24 may also have anamino acid sequence described in FIG. 1 or 3. The CD24 may exist in oneof two allelic forms, such that the C-terminal amino acid of the maturehuman CD24 may be a valine or an alanine. The C-terminal valine oralanine may be immunogenic and may be omitted from the CD24 to reduceits immunogenicity. The difference between the two alleles may affectthe risk of autoimmune diseases, including multiple sclerosis and RA.Nevertheless, since the two allelic forms affect the expression levelsof membrane-bounded form, the variation should not affect the functionof CD24.

Despite considerable sequence variations in the amino acid sequence ofthe mature CD24 proteins from mouse and human, they are functionallyequivalents in interaction with the danger-associated molecular patterns(DAMP). Since host response to DAMP is considered important for thepathogenesis of RA, the mouse and human CD24 may be functionallyequivalent in treating RA. As a result of sequence conservation betweenmouse and human CD24 primarily in the C-terminus and in the abundance ofglycosylation sites, significant variations in the mature CD24 proteinsmay be tolerated in using the CD24 to treat RA, especially if thosevariations do not affect the conserved residues in the C-terminus or donot affect the glycosylation sites from either mouse or human CD24.

a. Fusion

The CD24 may be fused at its N- or C-terminal end to a portion of amammalian Ig protein, which may be human or mouse. The portion may be aFc region of the Ig protein. The Fc region may comprise the hinge regionand CH2 and CH3 domains of the Ig protein. The Ig protein may be humanIgG1, IgG2, IgG3, IgG4, IgM, or IgA. The Fc portion may comprise SEQ IDNO: 6. The Ig protein may also be IgM, and the Fc portion may comprisethe hinge region and CH3 and CH4 domains of IgM. The CD24 may also befused at its N- or C-terminus to a protein tag, which may be GST, His,or FLAG. Methods for making fusion proteins and purifying fusionproteins are well known in the art.

b. Production

The CD24 may be heavily glycosylated, and may be involved in functionsof CD24 such as costimulation and interaction with danger-associatedmolecular patterns. The CD24 may be prepared using a eukaryoticexpression system. The expression system may entail expression from avector in mammalian cells, such as Chinese Hamster Ovary (CHO) cells.The system may also be a viral vector, such as a replication-defectiveretroviral vector that may be used to infect eukaryotic cells. The CD24may also be produced from a stable cell line that expresses CD24 from avector or a portion of a vector that has been integrated into thecellular genome. The stable cell line may express CD24 from anintegrated replication-defective retroviral vector. The expressionsystem may be GPEx™.

3. METHOD OF TREATMENT

The CD24 may be used to treat rheumatoid arthritis. The CD24 may beadministered to a subject in need thereof. The subject may be a mammalsuch as a human.

a. Combined CD24 Therapy

The CD24 may be combined with another drug, such as a disease-modifyingantirheumatic drug (DMARD). The drug may be a nonsteriodanti-inflammatory drug (NSAID), which may be a propionic acidderivative, an acetic acid derivative, an enolic acid derivative, afenamic acid derivative, or a selective Cox2 inhibitor. The drug mayalso be a corticosteroid or Methotrexate. The drug may be a biologic,which may be a TNF-α antagonist such as an anti-TNF-α antibody or afusion protein that binds to TNF-α (Enbrel), an anti-CD20 mAb, anantagonist of costimulatory molecule CD80 and CD86 such as a monoclonalantibody or a fusion protein (CTLA4Ig) that binds to the two molecules,or an antagonist for a receptor of either IL-1 or IL-6. The CD24 and theother drug may be administrated together or sequentially.

b. Pharmaceutical Composition

The CD24 may be contained in a pharmaceutical composition, which maycomprise a solvent, which may keep the CD24 stable over an extendedperiod. The solvent may be PBS, which may keep the CD24 stable for atleast 36 months at −20° C. (−15˜−25° C.). The solvent may be capable ofaccommodating the CD24 in combination with the other drug.

c. Dosage

The dose to be used for human may ultimately be determined through aclinical trial to determine a dose with acceptable toxicity and clinicalefficacy. The initial clinical dose for human may be estimated throughpharmacokinetics and toxicity studies in rodents and non-human primates.The dose of CD24 may be 0.01 mg/kg to 1000 mg/Kg, and may be 1 to 500mg/kg, depending on the severity of disease being treated and the routeof administration.

d. Administration

The route of administration of the pharmaceutical composition may beparenteral. Parenteral administration includes, but is not limited to,intravenous, intraarterial, intraperitoneal, subcutaneous,intramuscular, intrathecal, intraarticular and direct injection intoaffected joints. For veterinary use, the agent may be administered as asuitably acceptable formulation in accordance with normal veterinarypractice. The veterinarian can readily determine the dosing regimen androute of administration that is most appropriate for a particularanimal. The pharmaceutical composition may be administered to a humanpatient, cat, dog, large animal, or an avian.

The CD24 may be administered simultaneously or metronomically with othertreatments. The term “simultaneous” or “simultaneously” as used herein,means that the CD24 and other treatment be administered within 48 hours,preferably 24 hours, more preferably 12 hours, yet more preferably 6hours, and most preferably 3 hours or less, of each other. The term“metronomically” as used herein means the administration of the agent attimes different from the other treatment and at a certain frequencyrelative to repeat administration.

The CD24 may be administered at any point prior to another treatmentincluding about 120 hr, 118 hr, 116 hr, 114 hr, 112 hr, 110 hr, 108 hr,106 hr, 104 hr, 102 hr, 100 hr, 98 hr, 96 hr, 94 hr, 92 hr, 90 hr, 88hr, 86 hr, 84 hr, 82 hr, 80 hr, 78 hr, 76 hr, 74 hr, 72 hr, 70 hr, 68hr, 66 hr, 64 hr, 62 hr, 60 hr, 58 hr, 56 hr, 54 hr, 52 hr, 50 hr, 48hr, 46 hr, 44 hr, 42 hr, 40 hr, 38 hr, 36 hr, 34 hr, 32 hr, 30 hr, 28hr, 26 hr, 24 hr, 22 hr, 20 hr, 18 hr, 16 hr, 14 hr, 12 hr, 10 hr, 8 hr,6 hr, 4 hr, 3 hr, 2 hr, 1 hr, 55 mins., 50 mins., 45 mins., 40 mins., 35mins., 30 mins., 25 mins., 20 mins., 15 mins, 10 mins, 9 mins, 8 mins, 7mins., 6 mins., 5 mins., 4 mins., 3 mins, 2 mins, and 1 mins. The CD24may be administered at any point prior to a second treatment of the CD24including about 120 hr, 118 hr, 116 hr, 114 hr, 112 hr, 110 hr, 108 hr,106 hr, 104 hr, 102 hr, 100 hr, 98 hr, 96 hr, 94 hr, 92 hr, 90 hr, 88hr, 86 hr, 84 hr, 82 hr, 80 hr, 78 hr, 76 hr, 74 hr, 72 hr, 70 hr, 68hr, 66 hr, 64 hr, 62 hr, 60 hr, 58 hr, 56 hr, 54 hr, 52 hr, 50 hr, 48hr, 46 hr, 44 hr, 42 hr, 40 hr, 38 hr, 36 hr, 34 hr, 32 hr, 30 hr, 28hr, 26 hr, 24 hr, 22 hr, 20 hr, 18 hr, 16 hr, 14 hr, 12 hr, 10 hr, 8 hr,6 hr, 4 hr, 3 hr, 2 hr, 1 hr, 55 mins., 50 mins., 45 mins., 40 mins., 35mins., 30 mins., 25 mins., 20 mins., 15 mins., 10 mins., 9 mins., 8mins., 7 mins., 6 mins., 5 mins., 4 mins., 3 mins, 2 mins, and 1 mins.

The CD24 may be administered at any point after another treatmentincluding about 1 min, 2 mins., 3 mins., 4 mins., 5 mins., 6 mins., 7mins., 8 mins., 9 mins., 10 mins., 15 mins., 20 mins., 25 mins., 30mins., 35 mins., 40 mins., 45 mins., 50 mins., 55 mins., 1 hr, 2 hr, 3hr, 4 hr, 6 hr, 8 hr, 10 hr, 12 hr, 14 hr, 16 hr, 18 hr, 20 hr, 22 hr,24 hr, 26 hr, 28 hr, 30 hr, 32 hr, 34 hr, 36 hr, 38 hr, 40 hr, 42 hr, 44hr, 46 hr, 48 hr, 50 hr, 52 hr, 54 hr, 56 hr, 58 hr, 60 hr, 62 hr, 64hr, 66 hr, 68 hr, 70 hr, 72 hr, 74 hr, 76 hr, 78 hr, 80 hr, 82 hr, 84hr, 86 hr, 88 hr, 90 hr, 92 hr, 94 hr, 96 hr, 98 hr, 100 hr, 102 hr, 104hr, 106 hr, 108 hr, 110 hr, 112 hr, 114 hr, 116 hr, 118 hr, and 120 hr.The CD24 may be administered at any point prior after a previous CD24treatment including about 120 hr, 118 hr, 116 hr, 114 hr, 112 hr, 110hr, 108 hr, 106 hr, 104 hr, 102 hr, 100 hr, 98 hr, 96 hr, 94 hr, 92 hr,90 hr, 88 hr, 86 hr, 84 hr, 82 hr, 80 hr, 78 hr, 76 hr, 74 hr, 72 hr, 70hr, 68 hr, 66 hr, 64 hr, 62 hr, 60 hr, 58 hr, 56 hr, 54 hr, 52 hr, 50hr, 48 hr, 46 hr, 44 hr, 42 hr, 40 hr, 38 hr, 36 hr, 34 hr, 32 hr, 30hr, 28 hr, 26 hr, 24 hr, 22 hr, 20 hr, 18 hr, 16 hr, 14 hr, 12 hr, 10hr, 8 hr, 6 hr, 4 hr, 3 hr, 2 hr, 1 hr, 55 mins., 50 mins., 45 mins., 40mins., 35 mins., 30 mins., 25 mins., 20 mins., 15 mins., 10 mins., 9mins., 8 mins., 7 mins., 6 mins., 5 mins., 4 mins., 3 mins, 2 mins, and1 mins.

The following examples are provided to illustrate the methods of theinvention and are by no means to limit the use of the methods.

Example 1 Soluble CD24 Proteins

The extracellular domain of CD24 was fused to IgG1 Fc. The amino acidcomposition of the CD24 fusion protein is provided in FIG. 1. Areplication-defective retroviral vector that drives expression of theCD24Ig fusion protein was then generated. The GPEx™ (an acronym for geneproduct expression) system offers several important advantages, the mostimportant of which is the, on average, >1000 insertions/cell but withonly 1 copy/insertion. Moreover, since the retrovirus preferentiallyinserts into the transcriptional active locus, the GPEx™ resulted in ahigh level of expression of the targeted protein. Stable cell lines thatproduce a high yield of CD24Ig were generated. In addition 45 grams ofGLP grade products and −100 grams of cGMP grade products were produced.The methods used for downstream processing of media harvested from thebioreactor are summarized in the flow chart below (FIG. 2).

Harvest Clarification

The bioreactor culture media was clarified using Cuno 60M02 Maximizerdepth filters followed by a Millipore Opticap 0.22 um filter. Thefiltrate was collected into a sterile collection bag. Samples wereobtained for CD24-Fc yield quantitation by ELISA.

Protein A Capture

The clarified media was passed over a column of Protein A resin (GEHealthcare MabSelect) at a concentration not exceeding 16 g/L of resin(based on ELISA) and a contact time of 4 minutes. The column was washedwith the equilibration buffer (50 mM Tris+0.15M NaCl pH7.5), then with10 mM sodium citrate/citric acid pH 6.0 for 5cvs. Bound CD24Ig waseluted from the column using 10 mM sodium citrate/citric acid pH 3.5

Viral Inactivation

The Protein A eluate fraction was immediately brought to pH 3.0 with theaddition of 2M Hydrochloric acid and held at this pH for 30 minutes atambient temperature. It was then brought to pH 5.0 with the addition of1M Tris base, and filtered to clarity using a 0.65 um glass fiber filter(Sartorius Sartopure GF2) and 0.2 um (Sartorius Sartopore 2) into asterile collection bag.

SP-Sepharose Chromatography

The viral inactivated material was applied to a column of SP-Sepharose(GE Healthcare) at a concentration not exceeding 25 g/L of resin (basedon A280 nm of 1.22=1 mg/mL) and a linear flow rate of 250 cm/hr. Thecolumn was washed with the equilibration buffer (10 mM sodiumcitrate/citric acid pH 5.0) and bound CD24Ig was eluted from the columnusing 10 mM sodium citrate/citric acid+0.2M NaCl pH5.0. The effluent wascollected into a sterile collection bag.

Mustang Q Chromatography

The SP-Sepharose elute was adjusted to pH 7.5 by the addition of 1M Trisbase and diluted with WFI to reduce the conductivity. The dilutedmaterial was applied to a Mustang Q filter (Pall) at a concentration notexceeding 0.5 g/L of resin (based on A280 nm of 1.22=1 mg/mL) and at aflow rate of 5 column volumes/minute. The filter was washed with theequilibration buffer (10 mM Tris pH 7.5) and the CD24-Fc is contained inthe flow through and is collected into a sterile collection bag.

Viral Filtration

The Mustang Q flow through was then filtered at a constant pressure of30 psi through a 0.2 mM filter and a Millipore NFP viral filter (nominalpore size 20 nm) and was collected into a sterile collection bag.

Concentration and Final Formulation

The product was concentrated and diafiltered using a 10 kDaultrafiltration membrane (Millipore Prep/Scale) into a 10 mM sodiumphosphate, 150 mM sodium chloride pH 7.2 at approximately 10 mg/mL finalconcentration as determined by absorbance at 280 nm. Analytical sampleswere drawn from the bulk whilst in a biosafety cabinet. Labeling wasperformed and the samples were delivered to QC for testing while thebulk aliquots were stored at 2-8° C. pending release.

Viral Clearance Studies

The viral clearance validation was performed at Cardinal Health, NC, onsamples prepared at CHM. Qualified scientists from Gala Biotechperformed the chromatography and filtration steps in the Cardinal HealthViral Validation facility with the assistance of Cardinal Healthpersonnel. The scale down procedure was developed from the 200 L scaleprocess. Two viruses were chosen to be used in this study. The first wasXenotropic murine Leukemia virus (XMuLv), which is an enveloped RNAvirus of 80-130 nm in size from the Retroviridae viral family. Thesecond was Porcine Parvovirus (PPV), which is a nonenveloped DNA virusof 18-26 nm in size. This is considered a robust virus, and was expectedto demonstrate a much lower viral reduction through the purificationprotocol than the XMuLv.

Example 2 CD24 Pharmacokinetics

1 mg of CD24IgG1 (CD24Fc) was injected into naïve C57BL/6 mice andcollected blood samples at different timepoints (5 min, 1 hr, 4 hrs, 24hrs, 48 hrs, 7 days, 14 days and 21 days) with 3 mice in each timepoint.The sera were diluted 1:100 and the levels of CD24Ig was detected usinga sandwich ELISA using purified anti-human CD24 (3.3 μg/ml) as thecapturing antibody and peroxidase conjugated goat anti-human IgG Fc (5μg/ml) as the detecting antibodies. As shown in FIG. 4 a. The decaycurve of CD24Ig revealed a typical biphase decay of the protein. Thefirst biodistribution phase had a half life of 12.4 hours. The secondphase follows a model of first-order elimination from the centralcompartment. The half life for the second phase was 9.54 days, which issimilar to that of antibodies in vivo. These data suggest that thefusion protein is very stable in the blood stream. In another study inwhich the fusion protein was injected subcutaneously, an almostidentical half life of 9.52 days was observed (FIG. 4 b). Moreimportantly, while it took approximately 48 hours for the CD24Ig toreach peak levels in the blood, the total amount of the fusion proteinin the blood, as measured by AUC, was substantially the same by eitherroute of injection. Thus, from therapeutic point of view, differentroute of injection should not affect the therapeutic effect of the drug.This observation greatly simplified the experimental design for primatetoxicity and clinical trials.

Example 3 CD24 for Treating RA

For decades, it has been assumed that RA is predominantly a T-cellmediated autoimmune diseases. In the last two decades, there is areawaking on the possible role for antibodies and B lymphocytes in RApathogenesis. Thus, in addition or rheumatoid factors, a host ofautoreactive antibodies have been found in RA patients, although it hasnot been definitively addressed in human. However, several lines ofevidence have demonstrated that in the mouse models, antibodies specificfor either ubiquitous or tissue specific antigens are sufficient tocause RA symptoms. For instance, antibodies from the K/BxN TCRtransgenic mice were found to be fully capable of transferring RA-likediseases in the new host. Likewise, a cocktail for 4 anti-collagenantibodies is now widely used to induce RA in the mouse. This model isnow called CAIA, for collagen antibody-induced arthritis.

Genetic analyses of CAIA model indicate critical roles for complement.Although other possibilities exist, these requirements suggest potentialinvolvement of antibody-mediated tissue damage in the pathogenesis ofRA. The linkage between tissue damage and inflammation is along-standing observation in immunology. Nearly two decades ago,Matzinger proposed what was popularly called danger theory. In essence,she argued that the immune system is turned on when it senses thedangers in the host. Although the nature of danger was not well definedat the time, it has been determined that necrosis is associated with therelease of intracellular components such as HMGB1 and Heat-shockproteins, which were called DAMP, for danger-associated molecularpatterns. DAMP were found to promote production of inflammatorycytokines and autoimmune diseases. In animal models, inhibitors of HMGB1and HSP90 were found to ameliorate RA. The involvement of DAMP raisedthe prospect that negative regulation for host response to DAMP can beexplored for RA therapy.

CD24-Siglec 10 Interaction in Host Response to Tissue Injuries

Using acetaminophen-induced liver necrosis and ensuring inflammation, weobserved that through interaction Siglec G, CD24 provides a powerfulnegative regulation for host response to tissue injuries. CD24 is a GPIanchored molecules that is broadly expressed in hematopoietic cells andother tissue stem cells. Genetic analysis of a variety of autoimmunedisease in human, including multiple sclerosis, systemic lupuserythromatosus, RA, and giant cell arthritis, showed significantassociation between CD24 polymorphism and risk of autoimmune diseases.Siglec G is a member of I-lectin family, defined by their ability torecognize sialic acid containing structure. Siglec G recognized sialicacid containing structure on CD24 and negatively regulates production ofinflammatory cytokines by dendritic cells. In terms of its ability tointeract with CD24, human Siglec 10 and mouse Siglec G are functionallyequivalent. However, it is unclear if there is a one-to-one correlationbetween mouse and human homologues. Although the mechanism remains to befull elucidated, it is plausible that SiglecG-associated SHP1 may beinvolved in the negative regulation. These data, reported in Sciencerecently, leads to a new model in which CD24-Siglec G/10 interaction mayplay a critical in discrimination pathogen-associated molecular pattern(PAMP) from DAMP (FIG. 5).

At least two overlapping mechanisms may explain the function of CD24.First, by binding to a variety of DAMP, CD24 may trap the inflammatorystimuli to prevent their interaction with TLR or RAGE. This notion issupported by observations that CD24 is associated with several DAMPmolecules, including HSP70, 90, HMGB1 and nucleolin. Second, perhapsafter associated with DAMP, CD24 may stimulate signaling by Siglec G.Both mechanisms may act in concert as mice with targeted mutation ofeither gene mounted much stronger inflammatory response. In fact, DCcultured from bone marrow from either CD24−/− or Siglec G−/− miceproduced much higher inflammatory cytokines when stimulated with eitherHMGB1, HSP70, or HSP90. In contrast, no effect were found in theirresponse to PAMP, such as LPS and PolyI:C. These data not only provideda mechanism for the innate immune system to distinguish pathogen fromtissue injury, but also suggest that CD24 and Siglec G as potentialtherapeutic targets for diseases associated with tissue injuries.

Therapeutic Effect of CD24Fc on Collagen-Antibody-Induced Arthritis

Given the suspected role for innate immunity to tissue injury in thepathogenesis of RA and the role for CD24-Siglec G/10 pathway innegatively regulate such response, the possibility of stimulating thispathway to treat RA was explored. Pathogenesis of essentially allautoimmune diseases involves induction of immune response to autoantigenand autoimmune destruction. The autoimmune destructive phase was focusedon, based the novel function of CD24-Siglec G interaction. Therefore,for the preliminary analysis, collagen antibody-induced arthritis modelwas adopted to evaluate potential therapeutic effect.

As shown in FIG. 6 a, the CAIA was induced on 8 weeks old BALB/c mice byi.v. injection of a cocktail of 4 anti-collagen mAbs (MD Biosciences,St. Paul, Minn.) at 2 mg/mouse on day 1, and i.p. injection of 100μg/mouse of LPS (MD Bioscience) on day 3. The mice were treated on day 1with either 1 mg CD24Fc or equal volume of 1×PBS vehicle as negativecontrol. As shown in FIG. 6 b, in comparison with vehicle control,CD24Fc provided highly significant therapeutic effects.

To understand the mechanism by which CD24Fc reduces arthritis in thismodel, cytokines were measured from homogenized joints of CD24Fc treatedmice or PBS control group, and measured the supernatant of 200 μg tissuehomogenates by cytokine beads array. A typical example is shown in FIG.8 a, while the summary data are shown in FIG. 7 b. These datademonstrated that systematically administrated CD24 reduces the levelsof multiple inflammatory cytokines including TNF-α, IL-6, MCP-1(CCL2)and IL-1β.

The effect of CD24Fc is substantiated by histological analysis of thesynovial joints of CAIA mice, as presented in FIG. 8. On day 7 afterinduction of arthritis, H&E staining demonstrated that the jointsynoviums in the PBS group are heavily infiltrated with inflammatorycells including neutrophil, macrophage, and lymphocytes (FIG. 8 a). Thiswas much reduced in the CD24Fc treated mice (FIG. 8 b). In addition,sever cartilage damages were revealed by the loss of safranin O redstaining in PBS-treated (FIG. 8 c) mice, but not CD24Fc-treated group(FIG. 8 d).

To determine whether mice, CD24Fc have therapeutic effect on ongoing RA,treatment was started at either 5 or 7 days after induction of RA. Asshown in FIG. 9, significant reduction of RA score was observed as soonas two days after CD24Fc treatment. The therapeutic effect lasted forthe remaining period of observation even without additional treatment.These data further strengthen the therapeutic potential of CD24Fc onongoing diseases.

In order to estimate the therapeutic doses of CD24Fc in human, CD24Fcwas titrated through a wide range of doses. As shown in FIG. 10, aslittle as 2 microgram/mice is sufficient to have statisticallysignificant therapeutic effect.

Siglecg-Dependent Therapeutic Effect of CD24Fc

To determine whether CD24Fc protect mice by interacting with Siglec G,we determined if the therapeutic effect depends on the Siglecg gene.Since the Siglecg-deficient mice were produced with ES cells fromC57BL/6 mice, we used WT C57BL/6 mice as control. As shown in FIG. 11 a,since the B6 mice are known to be less susceptible to the CAIA, theoverall disease score is lower than that observed in the BALB/c mice.Nevertheless, a single injection of the CD24Fc essentially wiped out theclinical signs in the WT mice. Importantly, even though the disease isless severe in the Siglecg-deficient mice, CD24Fc had no therapeuticeffect. Therefore, the therapeutic effect of CD24Fc is strictlydependent on the Siglecg gene.

Taken together, the data described herein demonstrates high therapeuticefficacy of CD24Fc for CAIA. Given our extensive data on safety,stability and our successful manufacture of CD24Fc all point to greatpotential of the fusion protein as a therapeutic for RA.

Example 4 Toxicity and Pharmacokinetics of CD24

Toxicity

A study of CD24Fc in mice was performed in order to evaluate thetoxicity and systemic exposure of CD24Fc when intravenously administeredto mice once weekly for four weeks, and the reversibility of toxicityfollowing a four-week recovery period. A total of 320 male and femalemice were assigned to Groups 1-4 for evaluation of the toxicity(20/sex/group) and reversibility (20/sex/group). All of these mice weretreated intravenously with either PBS buffer or CD24Fc at doses of 12.5,35, or 125 mg/kg once weekly for 4 weeks. A total of 360 mice wereassigned to Groups 5-8 for evaluation of the systemic exposure of CD24Fcfollowing the first dose (3/sex in Group 5 and 27/sex/group in Groups6-8) and the last dose (6/sex in Group 5 and 30/sex/group in Groups6-8). All of these mice were treated intravenously with either PBSbuffer or CD24Fc at doses of 12.5, 35, or 125 mg/kg once on Day 1 oronce weekly for 4 weeks.

No morbidity or mortality was found in mice treated with either PBSbuffer or CD24Fc, with the exception that one male mouse treated with 35mg/kg of CD24Fc in Group 7 was found dead on Day 21. This death occurredin 1/680 of mice and did not appear to be associated with the treatment.No treatment-related changes were noted in clinical observations, bodyweights, food consumption, ophthalmology, hematology, coagulation,clinical chemistry, organ weights, and organ-to-body or to-brain weightratios. No treatment-related macroscopic and microscopic findings wereobserved in all examined animals.

Anti-CD24Fc antibody was detected in 10/30 of mice on Day 30 and 12/30of mice on Day 57, which appeared to be dose dependent with nodifference between male and female mice. A higher systemic exposurefollowing the first dose and last dose was observed in male animalscompared to female animals. An approximately linear increase in AUCvalues was observed when mice were treated with increasing doses ofCD24Fc from 12.5 mg/kg to 125 mg/kg. An accumulation of CD24Fc in micewas observed after dosing once weekly for four weeks.

In summary, no observed adverse effects were noted in mice treated withCD24Fc at doses of 12.5, 35 or 125 mg/kg once weekly for 4 weeks with a4 week recovery period. The NOAEL was considered to be equal to orgreater than 125 mg/kg.

Another study of CD24Fc was performed in cynomolgus monkeys to examinethe pharmacokinetics and potential toxicity, acquired immunity, andimmunogenicity following administration of CD24Fc administered byintravenous infusion once weekly for four weeks, and to evaluaterecovery from any effects of the test article over a dose-free period ofat least 10 weeks. The test article, CD24Fc (Lot No.FP004.12-06097-001), and the control article, 1× phosphate-bufferedsaline (PBS), pH 7.2 (Lot No. 654446), were supplied as a clear,colorless, preformulated aqueous solution and as a preformulated aqueoussolution, respectively. The test article formulation was used asreceived for dose administration.

Forty experimentally naive cynomolgus monkeys (20 males and 20 females),2.9 to 4.7 years of age for the males and 2.9 to 5.0 years of age forthe females, and weighing 2.1 to 3.2 kg for the males and 2.2 to 2.9 kgfor the females at the outset (Day −1) of the study, were assigned todose groups as shown in Table 2 below.

All animals were dosed via 1-hour intravenous infusion once weekly for 4consecutive weeks. The first day of dosing was designated Day 1 (04 and5 Sep. 2009, Sets A and B, respectively). The animals were evaluated forchanges in clinical signs (evaluations of morbidity and/or mortality[twice daily] and cage side observations [once daily], body weight(Weeks −2 and −1, and weekly thereafter), electrocardiography (prestudyand Day 28), ophthalmology (prestudy and Day 28), and clinical pathologyindices (including serum chemistry, hematology, and coagulation (threeprestudy and Days 28 and 98), and urinalysis [via cystocentesis on Days28 and 98]). Blood samples were collected for toxicokinetic analysispredose, 15 minutes, and 6, 24, and 72 hours in relation to doseadministration on Days 1 and 22, predose on Day 8, and on Day 29 (at thesame time of day as Day 22 end of infusion). Blood samples for flowcytometry were collected prestudy (three time points), and on Days 28and 98. Blood samples for anti-drug antibodies (ADA) were collectedprestudy and on Days 28 and 98. To evaluate the T-cell dependentantibody response (TDAR), the animals were immunized with a 1:1 emulsionof keyhole limpet hemocyanin (KLH) and Incomplete Freund's Adjuvant(IFA) injected intramuscularly on the thigh to achieve a dose of 750mg/animal on Day 10, and to recovery animals on Day 84. Blood samplesfor anti-KLH response were collected prestudy, and on Days 20, 24, 28,94, and 98. Twenty four (24) animals (3/sex/group) were euthanized oneweek after the last dose. The remaining 16 animals (2/sex/group) werecontinued on the study without further dosing, and euthanized on Day 98(76 days after the last dose). At termination, a full necropsy wasconducted on all animals, and tissues were collected, preserved,processed, and examined microscopically by a Study Pathologist certifiedby the American College of Veterinary Pathologists (ACVP). At recovery,a full necropsy was conducted on all animals, and tissues werecollected, preserved, processed, and selected tissues (based on clinicalpathology and gross observations) were examined microscopically by astudy pathologist certified by the ACVP.

The results showed that CD24Fc administered via 1-hour intravenousinfusion once weekly for 4 consecutive weeks at 12.5 mg/kg/week (Group2), 35 mg/kg/week (Group 3), or 125 mg/kg/week (Group 4) was generallywell tolerated in male and female cynomolgus monkeys through a 4-weekdosing period, followed by a 10 week recovery period. There were noCD24Fc-related changes in clinical observations, food consumption, bodyweights, electrocardiographic measurements, ophthalmic examinations,serum chemistry (one exception noted below), coagulation, urinalysisparameters, macroscopic findings, or organ weights or organ weightratios. There were no CD24Fc-related changes in peripheral bloodmononuclear cell subset counts obtained by flow cytometry, or anti-KLHimmunoglobulin G (IgG) responses to KLH challenge. There was a possibleapproximately 2-fold increase in ALP related to CD24Fc for one maleadministered 35 mg/kg/week at Day 98 compared to predose and Day 28values. There was also a possible CD24Fc-related slight decrease inhemoglobin at Day 28 for 35 mg/kg/week males. The magnitude and/ornature of these changes was not considered adverse in this study. On Day28, one male administered 35 mg/kg/week had an approximate 13% decreasein hemoglobin concentration. On Day 98 (76 days after the last dose),changes in hematology for this animal included a 21% decrease inhemoglobin, a 16% increase in red blood cell counts, a 21% decrease inmean cell volume (MCV), a 32% decrease in mean cell hemoglobin (MCH), a14% decrease in mean cell hemoglobin concentration (MCHC), a 24%increase in red cell distribution width (RDW), and an approximately2-fold increase in platelet and reticulocyte counts compared to prestudy(Day −2) levels. Day 98 blood smear analysis documented hypochromic redblood cells, microcytosis, anisocytosis, schistocytes, poikilocytes, andspherocytes, as well as an apparent increase in platelet numbers. Thesehematology changes are not considered typical findings in cynomolgusmonkeys. However, no corresponding histopathology abnormalities wereidentified, and these hematology findings were not observed in any otheranimals in the mid-dose (35 mg/kg/week) or high-dose (125 mg/kg/week)groups.

At Day 29 terminal necropsy, there were microscopic findings of fibrinthrombi in hepatic sinusoids and multifocal glial nodules in the brainof one female administered 125 mg/kg/week. The changes were mild andoccurred only in one animal, but they were not typical backgroundfindings. More extensive independent studies established that the lesionwas also observed in placebo samples and therefore was not drug-related.At recovery necropsy, histopathologic analysis of the following subsetof tissues was performed based on clinical pathology or gross findings:all tissues except for mammary glands from a male administered 35mg/kg/week, the duodenum from another male administered 35 mg/kg/week,and testes from one male administered 125 mg/kg/week. No microscopicfindings were found related to the administration of CD24Fc.

CD24Fc was detected in serum samples from all dosed animals. Nomeasurable amount of CD24Fc was detected in control or predose serumsamples. Incurred sample reanalysis (ISR) was performed, and the resultsmet the acceptance criteria. Serum was screened for the presence ofanti-CD24Fc antibody. Seven animals out of the 40 total were identifiedas positive at the prestudy time point (17.5%). This rate of positivesamples prior to test article administration is similar to the rateobserved in the validation (12.5%) which demonstrated a subpopulation ofanimals with pre-existing anti-CD24Fc antibodies. Three animals wereidentified as positive during the dosing period of the study. Thevalidation experiment also demonstrated that 10 μg/mL of CD24Fcinhibited the measurement of anti-CD24Fc antibodies in reference to thepositive control, and all dose groups at the Day 29 toxicokinetic timepoint had concentrations greater than 10 μg/mL measured in serum. Onlyone animal (125 mg/kg/week dose group) screened positive for anti-CD24Fcantibodies within the recovery interval.

Based on the hematology findings, the no-observed-adverse-effect level(NOAEL) for CD24Fc was considered to be 12.5 mg/kg/week under theconditions of this study when administered once weekly for four weeks.

Pharmacokinetics of CD24 in Mice and Monkeys

The pharmacokinetics of CD24Fc were examined in the mouse and cynomolgusmonkeys. These animal PK studies were carried out by two preclinicalcontract research organizations. The mouse study was performed by JOINNLaboratory at Beijing, China, while the cynomolgus monkey study wascarried out by Charles River Laboratory at Reno, Nev., USA. Both studieswere in compliance with the Good Laboratory Protocol (GLP) and the finalreports were audited. As an independent report, the WinNonLin analysisof pharmacokinetic characterizations of CD24Fc in this report wereperformed by either JOINN Laboratory at Beijing, China (PK in mouse) orOncolmmune Inc. (PK in cynomolgus monkey).

In the mouse PK study, CD24Fc was intravenously administered to miceonce weekly for four weeks. A total of 360 mice were assigned to Groups1-4 for evaluation of the systemic exposure of CD24Fc following thefirst dose (3/sex in Group 1 and 27/sex/group in Groups 2-4) and thelast dose (6/sex in Group 1 and 30/sex/group in Groups 2-4). All ofthese mice were treated intravenously with either PBS buffer or CD24Fcat doses of 12.5, 35, or 125 mg/kg once weekly for 4 weeks. Bloodsamples were collected at each time point from 3 mice/sex/group inGroups 1-4 via orbital vein for determination of the serumconcentrations of CD24Fc. For Group 1, blood samples were collectedprior to dosing on Days 1 and 29 and at 4 hours after dosing on Day 29.For Groups 2-4, blood samples were collected at 5 and 15 minutes; and 1,4, 8, 24, 48, 72, and 168 hours following dosing on Days 1 and 29; and366 hours following dosing on Day 29.

The summarized results of the mouse serum concentration of CD24Fcfollowing the first dose and last dose are included in Table 1, and theWinNonlin analysis of serum CD24Fc concentration vs. time points isincluded in FIG. 17.

TABLE 1 Pharmacokinetic parameters of CD24Fc in mice Female Male unit125 mg/kg 35 mg/kg 12.5 mg/kg 125 mg/kg 35 mg/kg 12.5 mg/kg First doseparameters T_(1/2) h 46.36 59.18 63.93 48.51 70.57 63.00 C_(max) mg/ml3.17 0.66 0.32 2.13 0.99 0.54 AUC_((0-168 h)) h*mg/ml 39.04 8.65 5.8544.00 19.99 7.55 AUC_(inf) h*mg/ml 43.53 11.84 8.00 49.10 26.13 10.18 Vdml/kg 192.06 252.33 144.09 178.18 136.39 111.55 Cl ml/h/kg 2.87 2.961.56 2.55 1.34 1.23 MRT h 39.08 67.85 56.68 39.58 61.09 66.21 C_(max)ratio 10.04 2.09 1.00 3.92 1.83 1.00 AUC ratio 5.44 1.48 1.00 4.82 2.571.00 Last dose parameters t_(1/2) h 87.51 131.86 128.63 75.02 167.2758.90 C_(max) mg/ml 3.61 0.40 0.14 5.07 0.76 0.65 AUC_((0-336 h))h*mg/ml 177.00 21.35 7.68 251.18 88.20 35.75 AUC_(inf) h*mg/ml 187.9526.41 9.07 258.93 112.49 36.36 Vd ml/kg 83.96 252.16 255.74 52.25 75.0829.22 Cl ml/h/kg 0.67 1.33 1.38 0.48 0.31 0.34 MRT h 91.64 121.30 106.0077.98 106.29 70.30 C_(max) ratio 25.09 2.81 1.00 7.86 1.17 1.00 AUCratio 20.72 2.91 1.00 7.12 3.09 1.00 Faccu 4.32 2.23 1.13 5.27 4.31 3.57

The summarized results of the monkey serum concentrations of CD24Fcfollowing the single dose are included in Table 2, and the WinNonlinanalysis of serum CD24Fc concentration vs. time points is included inFIG. 18.

TABLE 2 Pharmacokinetic parameters of CD24Fc in cynomolgus monkeys PKParameters unit 35 mg/kg 12.5 mg/kg t½ h 148.39 188.29 Cmax mg/ml 0.890.25 AUC(0-t) h * mg/ml 67.09 17.02 AUCinf h * mg/ml 67.31 20.06 Vdml/kg 108.21 153.10 Cl ml/h/kg 0.52 0.62 MRT h 204.22 151.07 Cmax ratio3.56 1.00 AUC ratio 3.36 1.00

Example 5 A Variant CD24 has Improved Activity Over Wild-Type CD24

This example shows that a CD24 polypeptide containing human CD24 missingthe polymorphic amino acid at position 57 (SEQ ID NO: 1) (CD24Fc) ismore effective for treating RA than a CD24 polypeptide containingwild-type CD24 (SEQ ID NO: 2) (CD24^(v)Fc).

Methods

Antibodies, Fusion Proteins and Other Materials

CD24Fc and CD24vFc were manufactured by OncoImmune, Inc.; Bovine type IIcollagen, Catalog No. 20022, Chondrex Inc., Redmond, Wash.; a cocktailof 4 anti-collagen mAbs Catolog No. CIA-MAB-50 for BALB/c and CIA-MAB-2Cfor C57BL/6 mice, MD Bioproducts, St. Paul, Minn.; Chick type IIcollagen, Catalog No. 20011, Chondrex Inc., Redmond, Wash.; CompleteFreund's adjuvant: Catalog No. 7008, Chondrex Inc., Redmond, Wash., withheat-killed M. tuberculosis H37 Ra (non-viable) at concentration of 1mg/ml; Complete Freund's adjuvant: Catalog No. 7023, Chondrex Inc.,Redmond, Wash., with heat-killed M. tuberculosis H37 Ra (non-viable) atconcentration of 5 mg/ml; Incomplete Freund's adjuvant: Catalog No.7002, Chondrex Inc., Redmond, Wash.; Lipopolysaccharide (LPS), CatalogNo. 9028, Chondrex Inc., Redmond, Wash., from E. coli 0111:B4;Cytometric Bead Array (CBA) Mouse Inflammation Kit, Catalog No. 552364,BD Biosciences, San Jose, Calif.; Cytometric Bead Array (CBA) HumanInflammatory Cytokines Kit, Catalog No. 551811, BD Biosciences, SanJose, Calif.

Experimental Animals

BALB/cAnNCr (01B05, NCI) and C57BL/6NCr (01055, NCI) mice, male, 7 weeksold, were purchased from the National Cancer Institute (NCI) atFrederick, Md. DBA/1J (000670, JAX) mice, male, 7 weeks old, werereceived from Jackson Laboratories. All mice were quarantined for 7 daysprior to immunization. During quarantine, the animals were examined forgeneral health and acceptability for use in this study. Individualanimals were identified by ear mark. Animal cages were identified bystudy number, animal number, and group number. To minimize cagevariation, different treatments were given to individual mouse in thesame cages and scored in a double-blind protocol.

CAIA Model

BALB/c mice (8 weeks old) received mAbs (2 mg/mouse) on day 1 inconjunction with either vehicle or fusion proteins. Mice received LPS(100 μg/mouse) on day 3, and were observed daily for 3 weeks. The fusionproteins (0.2 or 1 mg/mouse) or vehicles were injected once on day 1. Inthe C57BL/6 mice, the dose of anti-collagen antibodies was either 2mg/mouse or 4 mg/mouse.

CIA Models

CIA in DBA/1 mice. On day 1, 8-week old DBA/1 mice were immunized with100 μL of collagen-CFA emulsion (made by mixing 2 mg/ml of bovine typeII collagen with equal volume of CFA containing 1 mg/ml of M.tuberculosis) subcutaneously at the base of the tail. On day 10, micewere booster-immunized with 60 μL of collagen-IFA emulsion (made bymixing 2 mg/ml of collagen with equal volume of IFA) subcutaneously 1.5cm from the tail base. Treatments were initiated either before or afterthe development of symptom of arthritis.

CIA in C57BL/6 mice. On day 1, 8-week old C57BL/6 mice were immunizedwith 100 μL of collagen-CFA emulsion (made by mixing 4 mg/ml of chicktype II collagen with equal volume of CFA containing 5 mg/ml of M.tuberculosis) intradermally at the base of the tail. On day 21, boosterimmunization with the same collagen-CFA emulsion was administeredintradermally 1.5 cm from the tail base. On day 28, mice with clinicalsymptoms were randomized to receive either vehicle or CD24Fc.

Treatment in RA Models

The scoring of arthritis was based on the following scale.

0, normal; 1, mild, but definite redness and swelling of the ankle orwrist, or apparent redness and swelling limited to individual digits,regardless of the number of affected digits; 2, moderate redness andswelling of ankle of wrist; 3, severe redness and swelling of the entirepaw including digits; 4, maximally inflamed limb with involvement ofmultiple joints. A combined score of 4 limbs in a mouse was reported asthe disease score for the mouse.

Prophylactic treatment in CAIA was initiated at the same time as theanti-collagen antibodies.

Prophylactic model in CIA model in DBA/1 mice: On day 17, the immunizedmice were randomly divided into two groups and were treated with vehicle(PBS) or given doses of CD24Fc. The mice were observed double blind forthree weeks.

Therapeutic CIA in DBA/1 model with ongoing diseases were initiated ateither 25 days after the first immunization using mice with clinicalscores from 3 to 8.

Therapeutic CIA in the C57BL/6 mice was initiated on day 28 or usingonly those mice with a clinical score from 3 to 8.

Statistical Methods

Group means and standard deviation values (when deemed appropriate) werecalculated for all numerical data obtained. The difference betweenCD24Fc and control mice was statistically analyzed with Fisher PLSDTest, or t-test for pairwise comparisons.

Results

Therapeutic Effect of Non-Polymorphic CD24Fc on Collage-Antibody-InducedArthritis

Human mature CD24 exists in two allelic forms, in which either a valineor alanine is present at the C-terminus (position 57 of the CD24 aminoacid sequence). Fusion proteins including either allelic form mayprovoke anti-drug antibodies in some RA patients. Thus, in order toavoid immunogenicity, it was tested whether the polymorphic residue canbe removed from CD24, while maintaining regulatory function of CD24. Asdiagrammed in FIG. 12A, two fusion proteins were created, one with theentire extracellular domain of CD24^(v) allele (CD24^(v)Fc) (the matureCD24 sequence having SEQ ID NO: 2), while the other had a one amino aciddeletion at the C-terminus of the resulting mature CD24 (CD24Fc) (themature CD24 sequence having SEQ ID NO: 1). Both forms were expressed andpurified to a similar degree (FIG. 12B).

To determine whether the valine on CD24 is required for CD24-Siglec 10interaction, their binding of CD24Fc and CD24^(v)Fc to a Siglec 10Fcfusion protein was compared. As shown in FIG. 12C, CD24Fc interactedwith Siglec 10Fc in a dose-dependent manner. The interaction depended onsialic acid on CD24Fc as pre-treatment of CD24Fc with sialidaseprevented the binding. Surprisingly, while CD24^(v)Fc also interactedwith Siglec 10Fc, the interaction was significantly weaker than that ofCD24-Fc-Siglec 10Fc. The CAIA was induced in 8 week-old BALB/c mice byi.v. injection of a cocktail of 4 anti-collagen mAbs in conjunction withCD24^(v)Fc, CD24Fc, human IgG1Fc or equal volume of 1×PBS vehicle asnegative control. As shown in FIG. 12D, in comparison with vehiclecontrol, CD24Fc provided highly significant therapeutic effects.Surprisingly, CD24^(v)Fc was far less effective, with activity not verydifferent from the negative control. In experiments similar to the onesshown in FIG. 12D, FIGS. 12E and 12F also show that CD24Fc is moreeffective than CD24^(v)Fc, although unlike in the experiments in FIG.12D, CD24^(v)Fc did have more activity than the negative control.Comparisons of the therapeutic effects of CD24Fc and CD24^(v)FC indicatethat deleting the polymorphic amino acid residue is not only likely toremove a potential issue of immunogenicity, but it also significantlyincreases the anti-inflammatory activity of the CD24 protein. Since theFc portion was identical in the two constructs, the therapeutic effectis largely attributable to CD24 function. Since Fc protein oftenexacerbated arthritis (data not shown), vehicle controls were used forin vivo studies to avoid inducing a confounding effect.

Therapeutic Effect of CD24Fc in Collagen-Induced Arthritis (CIA) Models

Since CAIA primarily reflects joint inflammation initiated byantibody-induced tissue injuries, and since RA involves both adaptiveand innate immune-mediated destruction that can be better reflected inCIA setting, two CIA models were used to study to potential therapeuticeffect of the CD24Fc. First, the prophylactic effect of CD24Fc weretested in the DBA/1 mouse. As shown in FIG. 13A, treatment with a singledose of CD24Fc prior to the development of clinical symptomssubstantially reduced subsequent disease scores (P=0.02). To determinewhether CD24Fc confers therapeutic effect for ongoing CIA in the DBA/1mice, the treatment was initiated when the mice had arthritis scoresfrom 3 to 8. The CD24Fc (200 μg/mouse) or vehicle was delivered everyother day for 5 times. As shown in FIG. 13B, a clear reduction ofarthritis score was observed as early as after two treatments.Significant reduction of clinical symptoms was observed in the CD24Fcgroup (P=0.02).

It has been reported that chicken collagen induces severe arthritis inC57BL/6 background. Therefore, this model was used to substantiate thetherapeutic effect of CD24Fc. Since a significant variation in diseasescore was observed within the same group, 50% and 80% reductions ofdisease scores were used as the endpoints of the study. The percentageof mice that reached either therapeutic endpoint over a three weekperiod was compared. As shown in FIG. 14A, CD24Fc accelerated recoveryof mice after severe clinical signs had developed. To test thetherapeutic effect at the peak of diseases, another week was allowed topass for mice to reach peak clinical score, at which point mice weretreated with repeated injections of either 100 or 50 μg/mouse (onceevery other day for a total of 5 injections). As shown in FIG. 14B, atransient increase of recovery was achieved with 50 μg/mouse/injection.However, a sustained recovery was achieved with only 100μg/mouse/injection. These data demonstrate a dose-dependent therapeuticeffect even when the drug was administrated at the peak of diseases.

CD24Fc Inhibits Production of Inflammatory Cytokines by a HumanMacrophage Cell Line with shRNA Silencing of CD24

To determine whether CD24 regulates production of inflammatory cytokinesin human cell line, CD24 was silenced in human THP1 cell line and thendifferentiation into macrophage was induced by treating the cells withPMA. As shown in FIG. 15A, CD24 silencing substantially increasedproduction of TNFα, IL-1β, and IL-6. These data demonstrate an essentialrole for endogenous human CD24 in production of inflammatory cytokines.Importantly, CD24Fc strongly inhibited production of TNFα, as well asIL-1β and IL-6 (FIG. 15B). Consistent with the therapeutic effect invivo, CD24^(v)Fc was approximately 10-fold less effective in inhibitingthe production of inflammatory cytokines in the macrophage cell line(FIG. 15C).

CD24Fc Confers Protection by Signaling Through Siglec G

It has been reported that CD24Fc interacts with Siglec G in mice andSiglec 10 in humans. To determine whether CD24Fc signals through SiglecG, spleen cells from CD24^(−/−) mice were incubated with either vehicle,Fc or CD24Fc for 30 min, and tyrosine phosphorylation and SHP-1 bindingto Siglec G were measured. As shown in FIG. 16A, CD24Fc stronglystimulated tyrosine phosphorylation of Siglec G. Correspondingly, theamount of SHP1 co-precipitated with Siglec G was dramatically increased.These results demonstrate that CD24Fc is capable of signaling throughSiglec G.

To test the significance of CD24Fc signaling through Siglec G in therapyof RA, WT mice and Siglec G-deficient mice were compared for theirresponse to CD24Fc. As shown in FIG. 16B, when lower doses ofanti-collagen antibodies were used, disease was less severe, yet theprotection was completely dependent on the Siglecg gene. However, withincreased doses of anti-collagen antibodies, the protective effect wasonly partially dependent on Siglecg, as the protection was stillobvious, albeit less pronounced (52% in KO vs 72% in WT mice) (FIG.16C). These data demonstrate that while CD24Fc can signal through SiglecG to confer protection against CAIA, an additional mechanism exists thatallows CD24Fc to protect against RA in the absence of Siglec G.

DISCUSSION

Taken together, the results demonstrate that CD24Fc has potenttherapeutic effects in three mouse RA models, including a CAIA and twoCIA models. The efficacies in multiple models indicates that CD24Fc hasa therapeutic effect among RA in humans with different underlyingpathogenesis. For decades, it has been assumed that RA is predominantlya T-cell mediated autoimmune diseases. In the last two decades, therehas been a re-awaking on the possible role for antibodies and Blymphocytes in RA pathogenesis. Thus, in addition to rheumatoid factors,a host of auto-antibodies have been found in RA patients. Several linesof evidence have demonstrated that in the mouse models, antibodiesspecific for either ubiquitous or tissue specific antigens aresufficient to cause RA symptoms. For instance, antibodies from K/BxN TCRtransgenic mice were found to be fully capable of transferring RA-likediseases in the new host. Likewise, a cocktail of 4 anti-collagenantibodies is now widely used to induce RA in mice. Genetic analyses ofthe CAIA model indicate critical roles for complement. Although otherpossibilities exist, these requirements suggest potential involvement ofantibody-mediated tissue damage in the pathogenesis of RA. The efficacyof CD24Fc in this model demonstrates that the fusion protein may beuseful for antibody-mediated destruction phase in RA patients.

The CIA model is commonly used for RA as it can mimic both induction andeffector function of both adaptive and innate immunity. Two CIA modelswere used to validate the therapeutic effect of CD24Fc. Bovinecollagen-induced RA in DBA/1 mice is the most commonly used model. Theabove data show that CD24Fc reduced the disease score either before orafter onset of disease in this model. One drawback of the bovine CIAmodel is that only relatively small numbers of strains are susceptible.In particular, C57BL/6 mice, which are commonly used for genetic studiesare resistant. More recently, a protocol has been developed to induceCIA in C57BL/6 mice using chicken collagen. As shown above, CD24Fcaccelerated recovery of arthritis induced by chicken collagen. The factthat the CD24Fc confers protection in multiple models demonstrates therobustness of its therapeutic effect.

An important issue relating to drug development is mechanism of action.Since it has been reported that CD24Fc binds to both Siglec G and humanSiglec 10, the significance of this interaction was evaluated. In vitro,as shown above, CD24Fc signals through Siglec G and triggers tyrosinephosphorylation. In vivo, as shown above, CD24Fc works at least in partthrough Siglec G. These results demonstrate that it is plausible thatCD24Fc protects against RA through strengthening the Siglec G-mediatedprotection against innate immunity to DAMPs. To date, no RA drug hasbeen developed by fortifying the negative regulation over innateresponse to DAMPs. Therefore, CD24Fc and other fusion proteinscontaining variant CD24 missing the polymorphic A/V amino acid (SEQ IDNO: 1) represents a new class of therapeutics for RA. This approach maybe preferable to antibodies targeting individual DAMPs or inflammatorycytokins. Since multiple DAMPs are released during autoimmunedestruction, targeting individual DAMP may be less effective thantargeting a broad-spectrum regulator such as CD24-Siglec G pathway.Nevertheless, it should be pointed out that the data in this exampledemonstrate that the protection is not completely dependent on signalingthrough Siglec G. At least two additional mechanisms can be invoked.First, by binding to DAMPs, CD24 may reduce the amounts of DAMPsavailable for their agonist receptors, such as RAGE, TLR. Second, sinceCD24 is heterogeneously glycosylated, it may bind to other members ofSiglecs to confer negative regulation.

CONCLUSION

A fusion protein comprising a non-polymorphic extracellular domain ofhuman CD24 (comprising SEQ ID NO: 1) protects mice against arthritisinitiated by either anti-collagen antibodies or immunization ofcollagen. The protection is at least partially dependent on itsinteraction with Siglec G. The data demonstrate the potential ofharnessing the negative regulation of innate immunity to tissueinjuries. Unexpectedly, the non-polymorphic variant of CD24 is superiorto wild-type CD24 in suppressing inflammatory cytokine production andprotecting mice against RA.

1.-10. (canceled)
 11. A method of treating rheumatoid arthritis in asubject, comprising administering to a subject in need thereof a CD24protein comprising a mature human CD24, wherein the sequence of themature human CD24 comprises SEQ ID NO: 1, wherein the CD24 protein doesnot comprise an alanine or valine immediately C-terminal to SEQ IDNO:
 1. 12. The method of claim 11, wherein the CD24 protein furthercomprises a protein tag, wherein the protein tag is fused to theC-terminus of the mature human CD24 or at the N-terminus of the CD24protein.
 13. The method of claim 12, wherein the protein tag comprisesthe Fc portion of a mammalian Ig protein.
 14. The method of claim 13,wherein the Fc portion comprises (a) the hinge region and CH2 and CH3domains of the human Ig protein, wherein the Ig is selected from thegroup consisting of IgG1, IgG2, IgG3, IgG4, and IgA; or (b) the hingeregion and CH3 and CH4 domains of IgM.
 15. The method of claim 11,wherein the CD24 protein comprises SEQ ID NO:
 5. 16. The method of claim11, wherein the CD24 protein is soluble.
 17. The method of claim 11,wherein the CD24 protein is glycosylated.
 18. The method of claim 11,wherein the CD24 protein is produced using a eukaryotic proteinexpression system.
 19. The method of claim 18, wherein the expressionsystem comprises a vector contained in a Chinese Hamster Ovary cell lineor a replication-defective retroviral vector.
 20. The method of claim19, wherein the replication-defective retroviral vector is stablyintegrated into the genome of a eukaryotic cell.
 21. A method oftreating rheumatoid arthritis in a subject, comprising administering toa subject in need thereof a pharmaceutical composition comprising a CD24protein comprising a mature human CD24, wherein the sequence of themature human CD24 comprises SEQ ID NO: 1, wherein the CD24 protein doesnot comprise an alanine or valine immediately C-terminal to SEQ IDNO:
 1. 22. The method of claim 21, wherein the pharmaceuticalcomposition comprises a solvent.
 23. The method of claim 22, wherein thesolvent keeps the CD24 protein stable over an extended period of time.24. The method of claim 23, wherein the solvent is phosphate bufferedsaline (PBS).
 25. The method of claim 21, wherein the CD24 proteinfurther comprises a protein tag, wherein the protein tag is fused to theC-terminus of the mature human CD24 or at the N-terminus of the CD24protein.
 26. The method of claim 25, wherein the protein tag comprisesthe Fc portion of a mammalian Ig protein, wherein the Fc portioncomprises (a) the hinge region and CH2 and CH3 domains of the human Igprotein, wherein the Ig is selected from the group consisting of IgG1,IgG2, IgG3, IgG4, and IgA; or (b) the hinge region and CH3 and CH4domains of IgM.
 27. The method of claim 21, wherein the CD24 proteincomprises SEQ ID NO:
 5. 28. The method of claim 21, wherein the CD24protein is soluble.
 29. The method of claim 21, wherein the CD24 proteinis glycosylated.
 30. The method of claim 21, wherein the CD24 protein isproduced using a eukaryotic protein expression system.